Affinity-shifted probes for quantifying analyte polynucleotides

Abstract

Compositions, methods and devices for detecting and quantifying levels of an analyte polynucleotide in homogeneous assays using collections of soluble or immobilized hybridization probes. In certain preferred embodiments, the probes are immobilized in an array format. Polynucleotides may be quantified directly, or amplified in an in vitro nucleic acid amplification reaction prior to detection and quantitation. Amplification reactions may be performed in contact with the invented probes, and analyte amplicons quantified in real-time or end-point assays.

What is claimed is:  
     
         1 . A probe reagent for quantifying the amount of an analyte polynucleotide, comprising: 
 a first probe complementary to a first analyte sequence contained within the analyte polynucleotide, said first probe comprising a first oligonucleotide sequence; and    a second probe complementary to a second analyte sequence contained within the analyte polynucleotide, said second probe comprising a second oligonucleotide sequence, 
 wherein the first analyte sequence and the second analyte sequence are contiguous with each other and share at least one nucleotide position in common, and  
 wherein the first probe hybridizes to the analyte polynucleotide with a first affinity and the second probe hybridizes to the analyte polynucleotide with a second affinity, said first affinity and second affinity being different from each other.  
   
     
     
         2 . A probe reagent for quantifying the amount of an analyte polynucleotide, comprising: 
 an amount of a first probe complementary to a first analyte sequence contained within the analyte polynucleotide, said first probe comprising a first oligonucleotide sequence; and    an amount of a second probe complementary to a second analyte sequence contained within the analyte polynucleotide, said second probe comprising a second oligonucleotide sequence, 
 wherein the first probe hybridizes to the analyte polynucleotide with a first affinity and the second probe hybridizes to the analyte polynucleotide with a second affinity, said first affinity being greater than said second affinity, and  
 wherein said amount of said first probe is greater than or equal to said amount of said second probe.  
   
     
     
         3 . A probe reagent for quantifying the amount of an analyte polynucleotide, comprising: 
 an amount of a first probe complementary to a first analyte sequence contained within the analyte polynucleotide, said first probe having a first oligonucleotide sequence, and said first probe having a first specific activity; and    an amount of a second probe complementary to a second analyte sequence contained within the analyte polynucleotide, said second probe having a second oligonucleotide sequence, and said second probe having a second specific activity, 
 wherein the first probe hybridizes to the analyte polynucleotide with a first affinity and the second probe hybridizes to the analyte polynucleotide with a second affinity, said first affinity and second affinity being different from each other, and  
 wherein if the amount of the first probe is greater than or equal to the amount of the second probe, then the specific activity of the first probe is greater than or equal to the specific activity of the second probe.  
   
     
     
         4 . The probe reagent of any one of claims  1 ,  2  or  3 , wherein said first probe and said second probe are soluble probes.  
     
     
         5 . The probe reagent of any one of claims  1 ,  2  or  3 , wherein said first probe and said second probe are immobilized probes.  
     
     
         6 . The probe reagent of any one of claims  1 ,  2  or  3 , wherein either said first probe and said second probe are first and second soluble probes, or said first probe and said second probe are first and second immobilized probes.  
     
     
         7 . The probe reagent of  claim 6 , wherein the first oligonucleotide sequence comprises the complement of said first analyte sequence.  
     
     
         8 . The probe reagent of  claim 6 , wherein the first oligonucleotide sequence consists of the complement of said first analyte sequence.  
     
     
         9 . The probe reagent of  claim 6 , wherein said first oligonucleotide sequence and said second oligonucleotide sequence are identical to each other.  
     
     
         10 . The probe reagent of  claim 9 , wherein at least one of said first probe and said second probe comprises a nucleotide analog.  
     
     
         11 . The probe reagent of  claim 6 , wherein said first oligonucleotide sequence and said second oligonucleotide sequence are different from each other.  
     
     
         12 . The probe reagent of  claim 11 , wherein said first and said second probes are first and second self-reporting probes, each comprising a detectable label.  
     
     
         13 . The probe reagent of  claim 12 , wherein the detectable labels of the first and second self-reporting probes are identical detectable labels.  
     
     
         14 . The probe reagent of  claim 6 , wherein said first probe further comprises a first detectable label, and wherein said second probe further comprises a second detectable label.  
     
     
         15 . The probe reagent of  claim 14 , wherein said first detectable label and said second detectable label each comprise a chemiluminescent moiety.  
     
     
         16 . The probe reagent of  claim 14 , wherein said first detectable label and said second detectable label each comprise a fluorophore.  
     
     
         17 . The probe reagent of  claim 15 , wherein the chemiluminescent moiety of said first detectable label and the chemiluminescent moiety of the second detectable moiety are identical chemiluminescent moieties.  
     
     
         18 . The probe reagent of  claim 17 , wherein said identical chemiluminescent moieties comprise acridinium ester.  
     
     
         19 . The probe reagent of  claim 4 , wherein said first probe and said second probe are first and second linear probes.  
     
     
         20 . The probe reagent of  claim 5 , wherein said first probe and said second probe are first and second molecular beacon probes.  
     
     
         21 . The probe reagent of  claim 6 , wherein said first probe and said second probe are first and second self-reporting probes.  
     
     
         22 . The probe reagent of  claim 21 , wherein the first and second self-reporting probes are first and second molecular beacons.  
     
     
         23 . The probe reagent of  claim 22 , wherein the first and second molecular beacons each comprise a stem portion and a loop portion, and wherein said first and second molecular beacons differ from each other in the length of their respective stem portions.  
     
     
         24 . The probe reagent of  claim 22 , wherein the first and second molecular beacons each comprise a stem portion and a loop portion, and wherein said first and second molecular beacons differ from each other in the length of their respective loop portions.  
     
     
         25 . The probe reagent of  claim 22 , wherein at least one of said first and second molecular beacons comprises at least one nucleotide analog.  
     
     
         26 . A method of quantifying the amount of an analyte polynucleotide present in a test sample over a range extending from a lower limit amount to an upper limit amount, comprising the steps of: 
 (a) providing a probe reagent in accordance with any one of claims  1 ,  2  or  3 ;    (b) hybridizing said probe reagent to any of said analyte polynucleotide that may be present in said test sample;    (c) measuring a signal that indicates the magnitude of hybrid duplex formation in step (b); and    (d) quantifying from the signal measured in step (c) the amount of said analyte polynucleotide present in said test sample.    
     
     
         27 . The method of  claim 26 , wherein the first probe and the second probe of the probe reagent are both soluble probes.  
     
     
         28 . The method of  claim 26 , wherein the first probe and the second probe of the probe reagent are both immobilized probes.  
     
     
         29 . The method of  claim 26 , wherein the measuring step comprises measuring optically.  
     
     
         30 . The method of  claim 29 , wherein the measuring step comprises performing luminometry.  
     
     
         31 . The method of  claim 29 , wherein the measuring step comprises measuring by fluorometry.  
     
     
         32 . The method of  claim 26 , wherein the quantifying step comprises comparing the signal measured in step (c) to a standard curve.  
     
     
         33 . A method of preparing a probe reagent for quantifying an analyte polynucleotide, comprising the steps of: 
 (a) selecting a first oligonucleotide probe complementary to a first analyte sequence contained within the analyte polynucleotide, 
 wherein said first oligonucleotide probe hybridizes to the analyte polynucleotide with a first affinity to form a first duplex;  
   (b) selecting a second oligonucleotide probe complementary to a second analyte sequence contained within the analyte polynucleotide, 
 wherein said second oligonucleotide probe hybridizes to the analyte polynucleotide with a second affinity to form a second duplex, said second affinity being different from said first affinity, and  
 wherein the first analyte sequence and the second analyte sequence are contiguous with each other and share at least one nucleotide position in common; and  
   (c) combining the probes selected in steps (a) and (b), thereby preparing the probe reagent.    
     
     
         34 . The method of  claim 33 , wherein the first oligonucleotide probe in selecting step (a) comprises a first detectable label and the second oligonucleotide probe in selecting step (b) comprises a second label.  
     
     
         35 . The method of  claim 34 , wherein the first and second detectable labels are identical.  
     
     
         36 . The method of  claim 33 , further comprising a step for immobilizing the combined probes to a solid support.